1. Field of the Invention
The present invention relates to a method and an apparatus for counting megakaryocytes.
2. Description of the Related Art
In the field of clinical examination, significantly useful information for diagnosing diseases can be obtained by identifying and counting megakaryocytes. Usually, a constant number of megakaryocytes exist in the normal bone marrow. Megakaryocytes are mother cells of thrombocytes, and the number of megakaryocytes may fluctuate due to disease in some cases, for example, when there is a reduction or an increase in the number of thrombocytes in the peripheral blood. Accordingly, identifying and counting these megakaryocytes is useful for obtaining information for the presence of a disease. The number of megakaryocytes increases, for example, in idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), essential thrombocytopenia and chronic myelogenous leukemia, and the number of megakaryocytes decreases in aplastic anemia.
As for a conventional method for counting megakaryocytes, generally, bone marrow aspirate is appropriately dyed and the obtained sample is transferred to a hemocyte calculating board, such as a Fuchs-Rosenthal calculating board, so that the megakaryocytes can be observed under a microscope and identified and counted. Megakaryocytes are characterized by their form; by having large cells, polynucleic cells, and blurred-looking cytoplasm. However, the standard for recognizing megakaryocytes is very ambiguous, and additionally, the ratio of megakaryocytes in the bone marrow is very low. Therefore, determination of megakaryocytes through visual examination is highly inconsistent and differs depending on the measuring person.
In recent years, identifying and counting of megakaryocytes using the thesis of flow cytometer has been attempted. According to the document by Tomer et al (Tomer et al, Blood, 1988; 71: 1244-1252), for example, megakaryocytes are identified and counted by a two-color measuring method using fluorescence-labeled anti-platelet antibodies and propidium iodide. Concretely, first, mononuclear cells are separated from bone marrow aspirate. The obtained sample containing mononuclear cells contains megakaryocytes, and the megakaryocytes in this sample are labeled with fluorescence-labeled anti-platelet antibodies. Next, the cells in the sample are fixed with 2% of paraformaldehyde, and then, dyed with propidium iodide. Thus, the obtained sample is measured with a flow cytometer, so that the megakaryocytes can be identified and counted. According to this method, separation of molecular cells makes the operation complex, and measurement requires much time. In addition, there is a possibility that part of the megakaryocytes will not be collected during the separation operation, and thus, the megakaryocytes cannot be identified and counted precisely.
Meanwhile, US Patent Application Publication No. 2005-0003471 describes a method for measuring megakaryocytes using an automatic hemocyte counting apparatus. Concretely, first, Dami cells, which are a strain of megakaryocyte based cells, are cultivated so as to obtain a specimen containing megakaryocytes, or purified CD34 positive cells are cultivated so as to obtain a specimen containing megakaryocytes. Then, the obtained specimen is measured with an automatic hemocyte counting apparatus, and megakaryocytes appear in the obtained two-dimensional distributional diagram. In addition, the two-dimensional distribution diagram that is obtained by measuring a specimen containing purified megakaryocytes and the two-dimensional distribution diagram that is obtained by measuring a specimen that does not contain megakaryocytes are compared, and thereby, a region where megakaryocytes appear can be determined in the two-dimensional distribution diagram. However, various cells other than megakaryocytes are included in bone marrow aspirate that has been extracted from a living body. In particular, the regions where plasmacytes, which appear in bone marrow aspirate that has been extracted from a patient having multiple myeloma appear, are quite similar to those of megakaryocytes in the two-dimensional distribution diagram.
Therefore, a method by which megakaryocytes can be measured more precisely by separating megakaryocytes that are included in bone marrow aspirate from other cells, such as plasmacytes, has become required.